The Zakian lab has identified a new subfamily of DNA helicases, the Pif1p subfamily, conserved from yeast to humans. The subfamily is named for its prototype member, the Saccharomyces Pif1p helicase. Pif1p appears to be a regional specific helicase. Absence of Pif1p perturbs the replication of three different substrates: telomeric, mitochondrial and ribosomal DNA (rDNA). A second member of the Pif1p subfamily, the Saccharomyces Rrm3p affects the same three substrates but has opposite effects from Pif1p on each. Perturbed maintenance of these DNAs has been correlated with tumorigenesis as well as aging. We propose a combined genetic structural, and computational approach to understand the role of Pif1p-like helicases. Specifically, we wish to determine the functional significance of the sequence similarity that define the Pif1 subfamily. The genetic approach focuses on Rph1p, an essential protein from Schizosaccharomyces pombe that is 40% identical to both Pif1p and Rrm3p over a 455 amino acid region. Isolation and analysis of temperature sensitive alleles of rph1+ will be carried out with the goal of identifying its in vivo substrates. The S. pombe Rph1p and the S. cerevisiae Pif1p will be expressed in E. coli purified and analyzed biochemically to assess their enzymatic properties, with the ultimate goal of using x-ray crystallography to determine an atomic level structure for both proteins. Computational approaches will be used to identify signature motifs within the Pif1p subfamily that distinguish members of this subfamily from other helicases. Predictions that arise from structural and computational analysis will inform future genetic analyses.